This invention relates to a process for preparation of pharmaceutically acceptable salts of clavulanic acid, particularly but not exclusively alkali salts especially potassium clavulanate.
Clavulanic acid is the common name for (2R,5R,Z)-30(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. Clavulanic acid and its alkali metal salts and esters are active as inhibitors of beta lactamase produced by some Gram positive as well as Gram negative micro-organisms. In addition to inhibition of beta lactamase, clavulanic acid and alkali metal salts thereof also have a synergistic action with penicillin and cephalosporin antibiotics. Clavulanic acid and its salts are used in pharmaceutical preparations to prevent the deactivation of beta lactam antibiotics. Commercial preparations contain potassium clavulanate in combination with amoxycillin trihydrate. Potassium clavulanate is more stable than the free acid or other salts.
Clavulanic acid is prepared by fermentation of a micro-organism such as strains of Streptomyces such as S.clavuligerus NRRL 3585, S.jimonjinensis NRRL 5741 and S.katsurahamanus IFO 13716 and Streptomyces sp.P6621 FERM P2804. The aqueous culture obtained after fermentation is purified and concentrated in accordance with conventional processes for example filtration and chromatographic purification as disclosed in GB 1508977, prior to extraction of the aqueous solution with an organic solvent to obtain a solution of impure clavulanic acid in the solvent.
GB 1508977 discloses preparation of clavulanate salts by filtration of the fermentation broth by passage through an anionic exchange resin. This process may achieve acceptable yields but sophisticated chromatographic purification methods are required and the use of resin columns involves substantial investment for manufacture on a commercial scale.
GB 1543563 discloses a fermentation process wherein the pH value of the medium is maintained in the range 6.3 to 6.7. Pharmaceutically acceptable salts such as potassium clavulanate are prepared by re-salting from lithium clavulanate.
EP-A-0026044 discloses use of the tertiary butylamine salt of clavulanic acid as an intermediate for purification of clavulanic acid. This salt was known from BE862211 or DE 2733230 which disclosed that the salt was even more stable than the sodium or potassium clavulanate salts. Tertiary butylamine is a toxic compound and is also difficult to remove from waste water giving rise to serious pollution concerns.
EP-A-0562583 discloses use of salts of clavulanic acid with N,Nxe2x80x2-monosubstituted symmetric ethylene diamines such as N,Nxe2x80x2-diisopropyethylene diammonium diclavulanate as useful intermediates for isolation and preparation of pure clavulanic acid or alkaline metal clavulanate salts from ethyl acetate extract.
WO93/25557 discloses use of clavulanate salts with numerous amines as intermediates for preparation of clavulanic acid or pharmaceutically acceptable salts or esters.
EP-A-0594099 discloses use of tertiary octylamine with clavulanic acid as an intermediate in preparation of clavulanic acid or pharmaceutically acceptable salts.
WO94/21647 discloses use of N,Nxe2x80x2-substituted diamines such as N,Nxe2x80x2-diisopropylethylene diammonium diclavulanate as a useful intermediate for preparation of clavulanic acid and alkali salts.
WO94/22873 discloses use of novel tertiary diammonium salts of clavulanic acid such as N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-1,2-diaminoethane clavulanate as a useful intermediate for preparation of clavulanic acid and salts thereof.
The aim of this invention is to prepare clavulanic acid and its pharmaceutically acceptable salts, such as potassium clavulanate in a new and simple manner, wherein the desired substance is obtained in a high yield and of high purity.
According to the present invention a process for preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof, including the steps of:
a. adding an additional solvent to a solution of clavulanic acid in a water immiscible solvent;
b. contacting the solution with an amine;
c. isolating the amine salt of clavulanic acid formed; and
d. converting the amine salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof.
The clavulanic acid may be obtained by fermentation of a Streptomyces micro-organism such as Streptomyces sp.P6621 FERM P2804 as disclosed in JP Kokai 80-162993. Alternative Clavulanic acid producing Streptomyces strains such as S.Clavuligerus may be employed.
Solids may be removed from the fermentation broth by filtration or preferably by microfiltration.
Microfiltration of the broth may be carried out as disclosed in WO95/23870. In a preferred process according to this disclosure the aqueous fermentation broth containing crude clavulanic acid, mycelium, proteins and other suspended solid matter is purified by microfiltration at a pH value between 5.8 and 6.2 and a temperature about 20 to 40xc2x0 C. The purified filtrate may be concentrated by reverse osmosis and then directly extracted in a series of countercurrent centrifugal extractors with a water immiscible solvent, preferably ethyl acetate. The extraction is preferably carried out at a temperature between 15 to 25xc2x0 C. and a pH between 1 and 3. The extract is then dried to a water content below 0.1 mol. %, further concentrated by evaporation and decolorised with active charcoal to obtain a completely dry organic phase.
The concentration of crude clavulanic acid in the dry concentrated extract in the water immiscible solvent such as ethyl acetate may be between 8 g/l and 40 g/l, preferably between 20 g/l and 40 g/l.
Alternative water immiscible solvents include: methyl acetate, propyl acetate, n-butly acetate, methyl ethyl ketone, methyl isobutyl ketone and mixtures thereof.
The additional solvent may be selected from: alcohols, nitriles, ketones and mixtures thereof. Preferred additional solvent include C1 to C6 alcohols, acetonitrile acetone and mixtures thereof.
Especially preferred solvents are selected from: methanol, ethanol, isopropanol, n-butanol, isobutanol and acetonitrile. Methanol is the most preferred solvent.
The amine may be selected from tertiary butylamine, benzyl tertiary butylamine, tertiary octylamine adamantane amine and sec butylamine. Other amines which have been proposed for purification of clavulanic acid may be employed.
In a preferred embodiment of the invention the amine is of formula I 
wherein the substituents R1, R2, R3, and R4 are independently: hydrogen; C1 to C8 straight or branched alkyl; C2 to C4 hydroxyalkyl or wherein the groups NR1R2 and NR3R4 jointly denote a heterocyclic ring having 3 to 6 methylene groups bound to a nitrogen atom, one of these groups being optionally substituted with an oxygen or a sulphur atom or by an imino group; wherein R5 denotes hydrogen or methyl; and n is an integer from 1 to 3.
Especially preferred amines are symmetrical N,Nxe2x80x2-alkylene diamines, preferably N,Nxe2x80x2-diisopropylethylene diamine, N,Nxe2x80x2-diethylethylene diamine, N,Nxe2x80x2-dibenzylethylene diamine, N,N,Nxe2x80x2,Nxe2x80x2-tetramethylethylene diamine. N,Nxe2x80x2-diisopropylethylene diamine is especially preferred.
At least one equivalent of the selected alkylene diamine, preferably a 10% excess up to about 2 molar equivalents may be employed for preparation of the clavulanate ammonium salt.
In a preferred embodiment of the invention the reaction of clavulanic acid with N,Nxe2x80x2-monosubstituted symmetric diamines can be carried out in a mixture of ethyl acetate and methanol for example having 0 to 80% of methanol, preferably in the ratio 4:1 to 2:1, preferably 3:1.
Use of a mixture of ethyl acetate and methanol according to the present invention allows isolation of the diammonium diclavulanate salt free of undesired impurities such as clavam-2-carboxylate. The clavam-2-carboxylic acid is difficult to separate from clavulanic acid (see D Brown et al, JCS, Chem.Com. 1979, 282). According to the United States Pharmacopoeia US P23, page 385, 1995 no more than 0.01% of potassium clavam-2-carboxylate is permitted.
Surprisingly it has been discovered that the yield of the intermediary salts with N,Nxe2x80x2-monosubstituted symmetric diamines is substantially higher and is almost quantitative when the transformation is carried out in a mixture of solvents, preferably ethyl acetate and methanol, in comparison to the transformation carried out in a single solvent, for example as disclosed in U.S. Pat. No. 5,310,898.
The ammonium diclavulanate salts prepared in accordance with this invention may be used as intermediate compounds for preparation of completely pure clavulanic acid and its pharmaceutically acceptable salts such as potassium clavulanate. Akali metal clavulanate salts may be prepared using an appropriate source of alkali metal, for example potassium 2-ethyl hexanoate in isopropanol (the solvent containing between 0% and 4% water). Alternative alkali metal salts include alkali carbonates, bicarbonates, or hydroxides, organic carboxylic acids, alkanoic acid salts such as acetates, propionates, hexanoates of which potassium 2-ethyl hexanoate is especially preferred. Suitable salts in accordance with the present invention are pharmaceutically acceptable alkali salts such as the sodium, potassium, calcium and magnesium salts of which sodium and potassium, especially potassium are preferred. The invention is further described by means of example but in any limitative sense.